Bottom Line:
The clinical presentation associated with GRN mutations is heterogeneous and may include clinical probable Alzheimer's disease.The approximately 75% reduction in full-length GRN, suggests an unbalanced GRN metabolism in loss-of-function mutation carriers whereby more GRN is processed into granulins.We propose that plasma GRN levels could be used as a reliable and inexpensive tool to identify all GRN mutation carriers in early-onset dementia populations and asymptomatic at-risk individuals.

ABSTRACTMutations in the progranulin gene (GRN) are an important cause of frontotemporal lobar degeneration (FTLD) with ubiquitin and TAR DNA-binding protein 43 (TDP43)-positive pathology. The clinical presentation associated with GRN mutations is heterogeneous and may include clinical probable Alzheimer's disease. All GRN mutations identified thus far cause disease through a uniform disease mechanism, i.e. the loss of functional GRN or haploinsufficiency. To determine if expression of GRN in plasma could predict GRN mutation status and could be used as a biological marker, we optimized a GRN ELISA and studied plasma samples of a consecutive clinical FTLD series of 219 patients, 70 control individuals, 72 early-onset probable Alzheimer's disease patients and nine symptomatic and 18 asymptomatic relatives of GRN mutation families. All FTLD patients with GRN loss-of-function mutations showed significantly reduced levels of GRN in plasma to about one third of the levels observed in non-GRN carriers and control individuals (P < 0.001). No overlap in distributions of GRN levels was observed between the eight GRN loss-of-function mutation carriers (range: 53-94 ng/ml) and 191 non-GRN mutation carriers (range: 115-386 ng/ml). Similar low levels of GRN were identified in asymptomatic GRN mutation carriers. Importantly, ELISA analyses also identified one probable Alzheimer's disease patient (1.4%) carrying a loss-of-function mutation in GRN. Biochemical analyses further showed that the GRN ELISA only detects full-length GRN, no intermediate granulin fragments. This study demonstrates that using a GRN ELISA in plasma, pathogenic GRN mutations can be accurately detected in symptomatic and asymptomatic carriers. The approximately 75% reduction in full-length GRN, suggests an unbalanced GRN metabolism in loss-of-function mutation carriers whereby more GRN is processed into granulins. We propose that plasma GRN levels could be used as a reliable and inexpensive tool to identify all GRN mutation carriers in early-onset dementia populations and asymptomatic at-risk individuals.

Mentions:
We then determined whether the levels of GRN in plasma could be used to distinguish GRN loss-of-function mutation carriers from non-GRN carriers in our FTLD series. Using a GRN ELISA we analysed a subset of 207 patients with FTLD from our consecutive cohort with plasma availability, including seven loss-of-function GRN mutation carriers and patient NGR247 carrying the partial GRN deletion, p.A472_Q548del. A significant reduction in GRN levels was observed in all patients carrying GRN loss-of-function mutations to about one third of the level observed in non-GRN carriers (Fig. 1; Table 2). GRN levels ranged from 53 to 94 ng/ml (mean value ± SD: 68 ± 16 ng/ml) in mutation carriers, while non-GRN carriers showed levels from 115 to 386 ng/ml (mean value ± SD: 220 ± 47 ng/ml). Interestingly, GRN levels were variable among GRN mutation carriers with slightly higher expression in NGR019 (88 ng/ml) carrying the p.A9D mutation in the signal peptide sequence and NGR247 (94 ng/ml) carrying p.A472_Q548del, compared to the typical frameshift and nonsense mutation carriers (Fig. 2). Based on our data alone, any cut-off value between 94 and 115 ng/ml is associated with crude estimates of both sensitivity and specificity of 100%. By assuming normality of GRN levels after a square root transformation, we were able to explore continuous estimates of sensitivity and specificity with different cut-off values (Table 3). A cut-off value of 112 ng/ml maximized the average of the estimates of sensitivity and specificity. GRN levels in 70 control individuals ranged from 138 to 376 ng/ml (mean value ± SD: 228 ± 50 ng/ml) comparable to patients with FTLD without GRN mutations (Fig. 1). As expected, normal GRN expression (259 ng/ml) was observed in our MAPT mutation carrier.Figure 1

Mentions:
We then determined whether the levels of GRN in plasma could be used to distinguish GRN loss-of-function mutation carriers from non-GRN carriers in our FTLD series. Using a GRN ELISA we analysed a subset of 207 patients with FTLD from our consecutive cohort with plasma availability, including seven loss-of-function GRN mutation carriers and patient NGR247 carrying the partial GRN deletion, p.A472_Q548del. A significant reduction in GRN levels was observed in all patients carrying GRN loss-of-function mutations to about one third of the level observed in non-GRN carriers (Fig. 1; Table 2). GRN levels ranged from 53 to 94 ng/ml (mean value ± SD: 68 ± 16 ng/ml) in mutation carriers, while non-GRN carriers showed levels from 115 to 386 ng/ml (mean value ± SD: 220 ± 47 ng/ml). Interestingly, GRN levels were variable among GRN mutation carriers with slightly higher expression in NGR019 (88 ng/ml) carrying the p.A9D mutation in the signal peptide sequence and NGR247 (94 ng/ml) carrying p.A472_Q548del, compared to the typical frameshift and nonsense mutation carriers (Fig. 2). Based on our data alone, any cut-off value between 94 and 115 ng/ml is associated with crude estimates of both sensitivity and specificity of 100%. By assuming normality of GRN levels after a square root transformation, we were able to explore continuous estimates of sensitivity and specificity with different cut-off values (Table 3). A cut-off value of 112 ng/ml maximized the average of the estimates of sensitivity and specificity. GRN levels in 70 control individuals ranged from 138 to 376 ng/ml (mean value ± SD: 228 ± 50 ng/ml) comparable to patients with FTLD without GRN mutations (Fig. 1). As expected, normal GRN expression (259 ng/ml) was observed in our MAPT mutation carrier.Figure 1

Bottom Line:
The clinical presentation associated with GRN mutations is heterogeneous and may include clinical probable Alzheimer's disease.The approximately 75% reduction in full-length GRN, suggests an unbalanced GRN metabolism in loss-of-function mutation carriers whereby more GRN is processed into granulins.We propose that plasma GRN levels could be used as a reliable and inexpensive tool to identify all GRN mutation carriers in early-onset dementia populations and asymptomatic at-risk individuals.

ABSTRACTMutations in the progranulin gene (GRN) are an important cause of frontotemporal lobar degeneration (FTLD) with ubiquitin and TAR DNA-binding protein 43 (TDP43)-positive pathology. The clinical presentation associated with GRN mutations is heterogeneous and may include clinical probable Alzheimer's disease. All GRN mutations identified thus far cause disease through a uniform disease mechanism, i.e. the loss of functional GRN or haploinsufficiency. To determine if expression of GRN in plasma could predict GRN mutation status and could be used as a biological marker, we optimized a GRN ELISA and studied plasma samples of a consecutive clinical FTLD series of 219 patients, 70 control individuals, 72 early-onset probable Alzheimer's disease patients and nine symptomatic and 18 asymptomatic relatives of GRN mutation families. All FTLD patients with GRN loss-of-function mutations showed significantly reduced levels of GRN in plasma to about one third of the levels observed in non-GRN carriers and control individuals (P < 0.001). No overlap in distributions of GRN levels was observed between the eight GRN loss-of-function mutation carriers (range: 53-94 ng/ml) and 191 non-GRN mutation carriers (range: 115-386 ng/ml). Similar low levels of GRN were identified in asymptomatic GRN mutation carriers. Importantly, ELISA analyses also identified one probable Alzheimer's disease patient (1.4%) carrying a loss-of-function mutation in GRN. Biochemical analyses further showed that the GRN ELISA only detects full-length GRN, no intermediate granulin fragments. This study demonstrates that using a GRN ELISA in plasma, pathogenic GRN mutations can be accurately detected in symptomatic and asymptomatic carriers. The approximately 75% reduction in full-length GRN, suggests an unbalanced GRN metabolism in loss-of-function mutation carriers whereby more GRN is processed into granulins. We propose that plasma GRN levels could be used as a reliable and inexpensive tool to identify all GRN mutation carriers in early-onset dementia populations and asymptomatic at-risk individuals.